During human development, autonomic neurons align with and pattern alongside blood vessels. This patterning allows the autonomic nervous system to control the vascular function a phenomenon that is very useful during situations such as "fight or flight" responses where the blood vessels need to respond rapidly and involuntarily to stimuli. Since the alignment of blood vessels with autonomic neurons occurs during embryogenesis, human embryonic stem cells provide a system in which we can observe and understand how neurons and blood vessels differentiate and co-align to form a neurovascular unit.
We have developed a human embryonic stem cell differentiation model where we are able to visualize the early stages of human blood vessel and neuronal development and their co-alignment and patterning in real time over a period of three weeks. Using this model, we have identified a cell-adhesion protein T-Cadherin, present on both the blood vessels and neurons, which may act as the "molecular velcro" in attaching neuronal cells to the blood vessel networks. We have also identified small RNA molecules termed microRNAs that may regulate T-Cadherin protein expression during this process. In this context, we propose that the regulation of T-Cadherin expression by specific microRNAs leads to the differentiation of autonomic neurons and their co-patterning with the network of blood vessels. We will test this hypothesis in three distinct aims:
1. Is T-Cadherin expression necessary and sufficient to drive the formation of the neurovascular unit?
2. Does microRNA regulation of T-Cadherin expression affect neurovascular co-patterning?
3. Can we manipulate T-Cadherin expression to generate viable autonomic neurons from hES cells for possible therapeutic use?
A number of human disorders result from abnormal patterning or development of autonomic neurons. For example, Hirschsprung's disease in infants results from improper nerve development in the gut, leading to chronic bowel obstruction that necessitates immediate surgical intervention. Understanding how autonomic neurons emerge from their precursors and whether aligning with blood vessels are required for their differentiation is critical to develop regenerative therapies. Furthermore, understanding how proteins like T-Cadherin regulate fundamental interactions of different cell types such as blood vessels and neurons offers insights into organ development and tissue engineering.

Statement of Benefit to California:

During human development, specific types of neuronal cells termed 'autonomic neurons' align and pattern along with blood vessels. Autonomic neurons are part of the peripheral nervous system that controls many involuntary functions, including heart rate and blood pressure. Lack of proper autonomic neuronal development or degeneration of existing autonomic neurons can lead to human diseases such as Hirschsprung's disorder, in which improper development of autonomic neurons in the gut results in bowel obstruction. This research proposal aims to understand how blood vessels help the differentiation of autonomic neurons. To this end, our studies will employ a novel human embryonic stem cell differentiation model in which both neurons and blood vessels develop in the context of all three germ layers. The understanding gained from our proposed studies will broadly benefit several categories of patients in California, including those with autonomic nervous system disorders. Furthermore, gaining fundamental insight into neurovascular patterning using this model system will facilitate development of functional blood vessels with proper autonomic innervation for tissue engineering applications that will benefit large numbers of patients in California.
This project will also serve to accelerate innovation of human ES cell and microRNA therapeutics in California, to train more people in California to work on this cutting-edge technology, and to establish the foundation for the design of regenerative therapies for neurovascular disorders that will benefit a significant number of citizens of California.

Progress Report:

Year 1

The development of a blood vessel network that can respond to autonomic neuronal inputs is a critical achievement during organogenesis. This system confers a functional benefit by delegating the involuntary control of vascular function to the autonomic nervous system, a phenomenon that is best illustrated by the “fight or flight response”. During the past year, we have focused on understanding the requirement of a particular cell adhesion molecule for proper co-patterning between blood vessels and neurons. Using several in vitro models, we were able to observe the timecourse for the development of vascular tubes comprised of multiple cell types. We discovered that co-patterning between vascular endothelial cells and autonomic neurons requires the presence of third cell type, vascular smooth muscle cells. Furthermore, we have identified a critical cell adhesion molecule that facilitates the interaction between these three cell types. Our studies focused on how autonomic neurons emerge from their precursors and our finding that aligning with blood vessels helps their differentiation is highly relevant for the development of new regenerative therapies for diseases of the autonomic nervous system, and ultimately for the engineering of organs with functional vascular networks.

Year 2

The development of a blood vessel network that can respond to autonomic neuronal inputs is a critical proper organ formation and function. During the past year, we have focused on understanding the requirement of a particular cell adhesion molecule for proper co-patterning between blood vessels and neurons. In the past review cycle, we found that two different vascular cell types (endothelial cells and vascular smooth muscle cells) were required to drive stem cell differentiation towards a neuronal fate. In the past year, we showed that a direct cell-cell interaction between vascular smooth muscle and stem cells is required for their differentiation toward an autonomic fate. We found that this is due to a homotypic interaction between critical cell adhesion molecules expressed on the surface of both cell types. Understanding how autonomic neurons emerge from their precursors by aligning with blood vessels to drive their differentiation is highly relevant for the development of new regenerative therapies for diseases of the autonomic nervous system, and ultimately for the engineering of organs with functional vascular networks.

Year 3

Our overall goal was to understand how blood vessels and neurons come to be co-aligned in the human body, a process which we will refer to as “neurovascular co-patterning”.
Generally, stem cell research focuses on the derivation and differentiation of single lineages in isolation from other cell types. However, the true vertebrate body plan is not constructed in that way. Indeed, most biological processes – starting with the earliest stages of organogenesis in the embryo and extending to organ regeneration in the adult -- demand exquisitely coordinated co-patterning of multiple lineages (often derived from different embryonic germ layers) for function to be normal. Neurovascular co-patterning (i.e., alignment of nerves and blood vessels) is a prototype for such a developmental program. By examining neural and vascular co-development in a human embryonic stem cell model, we offer a mechanism by which such co-patterning might emerge and in which primitive blood vessels then play an active role in early neuronal specification and differentiation.
Neural crest cells are a type of stem cell that can differentiate (i.e., to become more specialized) into one of many neural or non-neural cell types. We provide evidence that neural crest cells respond to distinct cues from the cell types comprising blood vessels: endothelial cells and vascular smooth muscle cells. Specifically, these signals prompt the neural crest cells to specialize into a particular kind of neuronal cell called an autonomic neuron. These are the cell types that control basic body functions below the level of consciousness, such as the control of blood flow.
We discovered that the process of neurovascular co-patterning requires two independent events. First, the neural crest cells must respond to a factor produced and released by the blood vessels, called “nitric oxide”. This signal provides a molecular cue facilitating neural crest cell differentiation towards an autonomic fate, as opposed to all of the multiple non-neural fates to which it might also have been directed. Secondly, this process is then further refined by direct contact between neural crest cells and the cells lining the outside of each blood vessel (“vascular smooth muscle cells”). An interaction with a cell adhesion molecule present on both cell types (“T-cadherin”) solidifies the fate of the neural crest cells.
Understanding how this process occurs and which elements are required will allow us to develop a new method to generate autonomic neurons in culture, a still elusive goal of regenerative medicine. Further development of this strategy will serve a significant unmet medical need and facilitate development of reparative therapies for dysautonomias such as Hirschsprung’s disease.